Biological Features ＆ Preliminary Clinical Observation About PHA-CD3AK Cells

Because of the limitations of surgery, chemotherapy and radiotherapy, a new method to treat cancer is urgently needed. In recent years, along with molecular biology, rapid development has been made in the field of cell biology and biotechnology. The biotherapy techniques played increasingly important roles in combating cancer. Adoptive cellular immunotherapy (ACI) has become one of the research hotspot in recent years. Choosing ideal killer cells are the key to ACI.TIL, A-LAK, CTL, TAK, and CIK cells were discovered and used in clinical trials continuously. However, these different kinds of killer cells were found to have defects and shortcomings. In this study, a new adoptive immunotherapy cells, CD3AK cells, were discovered. This new killer cells are superior to other effector cells in proliferation multiplication, killing activity and induced time in vitro. This experimental study is the preliminary research of biological features and preliminary clinical observation of CD3AK cells. The major contents are as follows.1. A preliminary experimental study on inducement in vitro and biological features of PHA–CD3AK cells0bjective:To investigate the inducement method in vitro and the preliminary biological features of PHA–CD3AK cells in comparison with LAK cells. Method : We adopted anti-CD3 monoclonal antibody(anti-CD3mAb) ,recombinant human interleukin-2 (rhIL-2 ) and phytohemaglutinin (PHA) to induce jointly human peripheral blood mononucleus cells (PBMCs). FCM, LDH, and Giemsa's staining were used to assay the biological features of PHA-CD3AK cells, such as phenotype, killing activity and nuclear type. Results: PHA-CD3AK cells could be induced and proliferated by tracing anti-CD3mAb (0.05ug/mL) plus rhIL-2(300U/mL) and PHA(100ug/mL).The proliferation multiples and the time in vitro of high level proliferation multiple of PHA–CD3AK cells is longer than that of LAK cells. When the proportion between effector cells and target cells is 80:1, PHA-CD3AK cells can kill 56.5% of K562 cells. The cell phenotype ratio with CD3~+, CD4~+, and CD8~+of PHA–CD3AK cells were(86.5±5.89)% , (33.20±5.27)%, and (42.63±3.50)%, respectively. Karyotype of PHA- CD3AK cell is a normal diploid. The chromosome number is 46. Conclusions: PHA–CD3AK cells are a group of heterogeneous cells with lymphoblast-like features. PHA-CD3AK cells are normal liploid cells. PHA-CD3AK cells are easily not only to be induced and cloned in vitro, but also to induce long time in vitro and to enhance killing activity. It might be a promising tumor immunotherapy cell.2. Clinical observation of adoptive immunotherapy using autologous PHA–CD3AK cells in treatment of advanced malignant tumorObjective: To observe the therapeutic effects of adoptive immunotherapy using PHA–CD3AK cells induced by anti-CD3mAb in treatment of advanced malignant tumors.Methods:PHA,Anti-CD3mAb and rhIL-2 were added into peripheral blood mono-nuclear cells (PBMC) separated from peripheral blood of fifty-one patients with malignant tumor to induce and cultivate in vitro to make autologous PHA–CD3AK cells. Sample from the PHA–CD3AK cells were tested for quality control at twelve to fourteen days. The qualified autologous PHA–CD3AK cells were gathered and adjusted to the concentration of (4.0 - 6.0)×10~9/L.Then the qualified cells were used to treat fifty-one patients suffering from advanced malignant tumors. Every patient was treated for 3 courses. The cells were infused by intravenous for 5 times every course (1 time every 2 days), and the level of cell exceeded 1.0×10~9/L every time. And the total number of the cell exceeded 5×10~9/L every course. The level of T cell subsets, chemical analysis, the quality of life and adverse reaction of pre therapy were compared with data after the therapy. Results:The PHA-CD3AK cells met the quality standard of ideal cells, and can be done for the experimental treatment further. After treated with autologous PHA-CD3AK cells, the level of CD3~+, CD4~+, CD8~+ subsets and CD16~+ CD56~+ in the peripheral blood were significantly increased (P < 0.05); 45 patients were obviously improved in general symptoms. There was no abnormal change of chemical analysis and adverse reaction in all patients. In 51 patients, 6 patients reached CR, 14 patients reached PR, 10 patients reached MR, 12 patients reached SD, and 9 patients were still progressive. The total effective rate of autologous PHA–CD3AK cells was 58.8% and the clinical beneficial rate was 82.4%. The quality of life of the most patients was improved. Conclusion:The therapeutic effects of PHA–CD3AK cells in treatment of advanced malignant tumors were positive. Autologous PHA–CD3AK cells can improve immune function efficiently, with better safety and without adverse effect.The preliminary study of induced in vitro and biological features and clinical experimental practice on PHA-CD3AK supplies a lot of important experimental data and beneficial discovery. PHA-CD3AK cells might be safe, efficacious and promising tumor adoptive immunotherapy cells.